The Journal of Neuroscience, April 1, 1997, 17(7):2267–2272 Monoamine Oxidase Inhibition Causes a Long-Term Prolongation of the Dopamine-Induced Responses in Rat Midbrain Dopaminergic Cells Nicola B. Mercuri, Mariangela Scarponi, Antonello Bonci, Antonio Siniscalchi, and Giorgio Bernardi Clinica Neurologica, Dipartimento Sanita´ Pubblica, Universita´ di Roma Tor Vergata and Istituto Ricerca e Cura a Carattere Scientifico Ospedale Santa Lucia, Roma, Italy The way monoamine oxidase (MAO) modulates the depression over, the effects of DA were not largely prolonged during the of the firing rate and the hyperpolarization of the membrane simultaneous inhibition of MAO and the DA reuptake system. caused by dopamine (DA) on rat midbrain dopaminergic cells Interestingly, the actions of amphetamine were not clearly aug- was investigated by means of intracellular recordings in vitro. mented by MAO inhibition. The cellular responses to DA, attributable to the activation of From the present data it is concluded that the termination of somatodendritic D2/3 autoreceptors, were prolonged and did DA action in the brain is controlled mainly by MAO enzymes. not completely wash out after pharmacological blockade of This long-term prolongation of the dopaminergic responses both types (A and B) of MAO. On the contrary, depression of the suggests a substitutive therapeutic approach that uses MAO firing rate and membrane hyperpolarization induced by quinpi- inhibitors and DA precursors in DA-deficient disorders in which role (a direct D2 receptor agonist) were not affected by MAO continuous stimulation of the dopaminergic receptors is inhibition. Furthermore, although the inhibition of DA reuptake preferable. by cocaine and nomifensine caused a short-term prolongation of DA responses, the combined inhibition of MAO A and B Key words: pargyline; cocaine; nomifensine; intracellular re- enzymes caused a long-term prolongation of DA effects. More- cordings; substantia nigra; ventral tegmental area The time course of the action of dopamine (DA) on its receptors tive mechanism of termination of DA action in the brain occurs has been believed to be controlled primarily by the DA reuptake via its deamination by MAO. system. This assumption has been substantiated by extensive stud- ies demonstrating that the concentration of DA (Church et al., MATERIALS AND METHODS 1987; Di Chiara and Imperato, 1988; Galloway, 1988; Nomikos et Preparation of the tissue. The method used has been described previously al., 1990; Kalivas and Duffy, 1991) and the physiological effects of (Mercuri et al., 1995). In brief, Wistar rats (150–250 gm) were anesthe- tized with ether and killed. The brain was removed, and horizontal slices this catecholamine in the brain are enhanced by agents that are (thickness 300 mm) were cut by a vibratome starting from the ventral able to block its transporter (Einhorn et al., 1988; Williams and surface of the midbrain. In some experiments in which amphetamine was Lacey, 1989; Lacey et al., 1990; Mercuri et al., 1991a,b,c). It has used, coronal slices of the ventral mesencephalon were also cut (Lacey et been suggested, however, that the tone of DA is regulated not al., 1987). A single slice containing the substantia nigra and the ventral tegmental area (VTA) was then transferred into a recording chamber and only by the DA reuptake system but also by the DA synthesizing submerged completely in an artificial cerebrospinal fluid with a continu- and degrading enzymes. Accordingly, we have shown recently that ously flowing (2.5 ml/min) solution at 358C, pH 7.4. This solution con- stimulation of DA synthesis by levodopa (Mercuri et al., 1990) tained (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, and blockade of DA degradation by MAO inhibitors (MAOI) 11 glucose, 20 NaHCO3, gassed with 95% O2/5% CO2. (Mercuri et al., 1996) cause a DA-mediated depression of the Recordings. The recording electrodes (Clark 1.0–1.5 mm, thick wall), pulled by Narishige vertical and horizontal pullers, were filled with 2 M firing discharge of the dopaminergic neurons in the ventral KCl and had a tip resistance of 40–80 MV. The signals were obtained by mesencephalon. an amplifier (Axoclamp-2A, Axon Instruments, Foster City, CA) and To study possible changes in the DA-induced responses caused displayed on a pen recorder (Gould 2400 S) and on a digital oscilloscope by the inhibition of MAO enzymes, we made intracellular elec- (Tektronix) or saved on a tape recorder (Biologic) for off-line analysis. The tips of the electrodes were placed in the substantia nigra pars trophysiological recordings from dopaminergic neurons in the rat compacta (SNc) and VTA by using a dissecting microscope. mesencephalon maintained in vitro. Using the same preparation, Application of drugs. Drugs were made in stock solutions and bath- we also examined the effects of two DA reuptake inhibitors, applied at known concentrations via a three-way tap system. A complete cocaine and nomifensine, on responses to exogenously applied exchange of the solution in the recording chamber occurred in ;1 min. DA. Contrary to the common belief that the effects of DA are The following substances were used: DA hydrochloride; cocaine hy- drochloride; (1)-amphetamine sulfate; haloperidol; pargyline, which regulated mainly by the transporter, we found that a more effec- blocks both types of MOA (A and B) (Butcher et al., 1990), (Sigma, St. Louis, MO); quinpirole (LY 171555, Lilly); nomifensine (Hoechst- Received Dec. 6, 1996; revised Jan. 6, 1997; accepted Jan. 13, 1997. Roussel Pharmaceuticals, Frankfurt, Germany); clorgyline, which is more We thank G. Gattoni, M. Federici, and M. Tolu for their excellent technical selective for type A MAO (Johnston, 1968); and deprenyl, which is more assistance. selective for type B MAO (Knoll and Magyar, 1972) (Research Biomedi- Correspondence should be addressed to Dr. Nicola B. Mercuri, IRCCS, Santa cals, Natick, MA); and L-sulpiride (Ravizza). The changes in firing rate Lucia, Via Ardeatina, n. 306, 00179, Roma, Italy. induced by the drugs were normalized as a percentage of control (each Copyright q 1997 Society for Neuroscience 0270-6474/97/172267-06$05.00/0 neuron served as its own control). In some experiments the slices were 2268 J. Neurosci., April 1, 1997, 17(7):2267–2272 Mercuri et al. • Long-Term Prolongation of DA Responses Figure 1. Pargyline prolongation of DA responses. A, The bath application of DA (30 mM) produced a reversible hyperpolarization and inhibition of firing. The bar indicates the period of DA application. A long-term prolongation of the DA-induced hyperpolarization and inhibition (DA was applied for the same period of control) was observed when pargyline 10 mM was superfused on the cells for 30 min. The subsequent application of sulpiride (300 nM) antagonized the prolonged response to DA application. B, The inhibitory effects of quinpirole (100 nM) were not affected by the superfusion of pargyline (10 mM) for 30 min. Note that in this and the following figures the speed of the chart was changed at the points indicated (see x underneath) to show individual action potentials. Full amplitude of the action potential was not reproduced because of the limited frequency of the pen recorder. preincubated for 2–3 hr with pargyline (1–10 mM), clorgyline (1–30 mM), or deprenyl (1–10 mM) to allow sufficient time for steady-state MAO inhibition to develop (Harsing and Vizi, 1984), and then the neuronal responses to DA were evaluated. Data were expressed as mean 6 SEM. RESULTS Electrophysiological and pharmacological properties of DA cells The present results are based on intracellular recordings made from 120 spontaneous-firing midbrain dopaminergic cells in vitro. The properties of these “principal” neurons have been described (Llinas et al., 1984; Kita et al., 1986; Grace and Onn, 1989; Lacey et al., 1989; Johnson and North, 1992; Mercuri et al., 1995). They fired at a mean rate of 1.5 Hz, had a relatively long-lasting spike (.1.2 msec), and showed a voltage-dependent sag in membrane potential with hyper- polarizing pulses. A brief superfusion of DA (10–30 mM for 1–2 min) caused reversible membrane hyperpolarization and inhibition of spontaneous firing. When application of the solution containing DA was discontinued, the firing rate returned to basal value within 5–10 min (Figs. 1A, 2). A reversible inhibition of the spontaneous firing and a hyperpolarization were also observed when quinpirole (30 nM–1 MM for 30–60 sec) (n 5 7) or (1)amphetamine (10–20 mM) were bath-applied to these neurons (n 5 6) (Figs. 1B, 6). The cellular responses to DA and quinpirole application are attributable mainly to the activation of somatodendritic D2/D3 type receptors, which increases potassium conductance (Lacey et al., 1987). Furthermore, the membrane hyperpolarization and the depression of the sponta- neous discharge caused by amphetamine are attributable to the release of endogenous DA from calcium-insensitive stores (Mercuri Figure 2. Long-term prolongation of the DA-responses in pargyline and et al., 1989). effects of the combined perfusion of DA uptake blockers. a, Plot of the firing rate (percentage of control) versus time in control condition and The inhibition of MAO potentiates the effects of after a treatment with pargyline (10 mM). Note that pargyline produced an exogenously applied DA but not the effects enduring DA-induced firing inhibition. The bar illustrates the DA washout of quinpirole starting point. Note that the firing remained depressed even after 160 min After testing the effects of DA and quinpirole on the principal of DA washout. In b, the previous graph was superimposed with two other graphs to observe the different changes in DA responses caused by cells, these cells were then superfused with the nonspecific MAO pargyline alone or in combination with cocaine (3 mM) and nomifensine A and B inhibitor pargyline (1–100 mM for 20–50 min).